OBESITY Tighter lymphatic junctions prevent obesity Zippering of cellular junctions in intestinal lacteals prevents fat uptake

By Donald M. McDonald which is driven by longitudinal smooth compromised in metabolic syndrome and muscle cells in intestinal villi, can propel type 2 diabetes. In the absence of VEGF-B, ietary fats take a circuitous route fluid and cells in lymph at velocities up to VEGFR1 and NRP1 can act as decoy recep- from the intestine to the blood- 150 µm/s into downstream lymphatics en tors that compete for VEGF-A binding to stream, where they serve as nutri- route to the bloodstream (6). VEGFR2. In support of this idea, Zhang ents or are stored in adipose tissue. Zhang et al. sought to determine the et al. provide evidence that VEGFR1 and Fats processed in the digestive tract contributions of the endothelial cell re- NRP1 bind VEGF-A and prevent excessive are packaged by the intestinal epi- ceptors, vascular endothelial growth fac- VEGFR2 signaling that would otherwise Dthelium into tiny lipid-protein particles tor receptor–1 (VEGFR1) and neuropilin-1 promote button-to-zipper transformation called chylomicrons. Chylomicrons are (NRP1), to the regulation of fat transport. and thereby reduce chylomicron uptake taken up by lymphatic channels (lacteals) By deleting the corresponding genes, into lacteals. within villi that line the small intestine Vegfr1 (also known as Flt1) and Nrp1, in Zhang et al. add to the evidence that en- and are transported by lymphatic vessels endothelial cells of mice, they unexpect- dothelial cell junctions are dynamic struc- Downloaded from to the blood circulation. How chylomi- edly found that button junctions in lacte- tures. Button junction plasticity is evident crons enter lacteals has long been a mys- als transformed into zipper junctions and during development: Initial lymphatics tery, as the particles are too large to cross that this conversion was accompanied by have zipper junctions until late gestation, the endothelial cells that line lymphatics. resistance to obesity when mice were given when they change into button junctions Moreover, scattered openings observed in a high-fat diet (see the figure). through a process that requires the growth lacteals seemed too sparse to explain chy- This work was initiated to resolve a con- factor angiopoietin-2 (ANGPT2) (9, 10). Al- http://science.sciencemag.org/ lomicron entry, and evidence for vesicular troversy over the involvement of VEGFR1 ternatively, button junctions change into transport was limited (1, 2). The discovery and NRP1 in fat transport in obesity and zippers in growing lymphatics in inflamed of specialized, discontinuous button-like type 2 diabetes. According to one concept, tissues (9). This transformation is revers- junctions between lacteal endothelial cells VEGF-B, a ligand of VEGFR1 and NRP1, ible by treatment with the corticosteroid raised another possibility (3). Button junc- increases expression of fatty acid trans- dexamethasone (9). Further evidence of tions have open regions and closed regions porters in blood vessels through binding plasticity is provided by the dependence and are strikingly different from zipper and activating VEGFR1 and NRP1, which of lacteal function on VEGF-C, a ligand for junctions that tightly seal endothelial cells consequently promote fat transport into VEGFR3, and on delta-like ligand 4 (DLL4), in blood vessels and collecting lymphatics. tissues (7). A contrasting interpretation is a NOTCH ligand. Mice lacking Vegfc or

On page 599 of this issue, Zhang et al. (4) that VEGF-B improves vascular and meta- Dll4 have reduced dietary lipid absorp- on August 9, 2018 show that chylomicron entry is dependent bolic health by displacing another ligand, tion and are resistant to obesity (11, 12). on button junctions. Importantly, experi- VEGF-A, which can then activate VEGFR2 Genetic inactivation of Dll4 in lymphatic

) mental manipulations that led to transfor- signaling (8). VEGFR2 signaling is essential endothelial cells results in transformation 3 ( .

L mation of button junctions into zippers for maintaining vascular function, which is of button junctions into zippers (12). In T A E prevented chylomicron uptake and pro- U K

A L tected mice from developing obesity while on a high-fat diet. OM B Lymphatic Intestinal lymphatic vessels not only endothelial cell Button junctions S I ON F R transport chylomicrons but are also routes VEGF-A low, chylomicron uupptakake for the recirculation of fluid into blood. junctions Chylomicron According to the conventional view, fluid Button junctions between lacteal endothelial cells W I T H PE R MI S flows into lacteals along hydrostatic pres- allow chylomicron uptake. C E D sure gradients when their endothelial cells U When VEGF-A is available, O D are pulled apart by interstitial forces (5). Openings through loss of the decoy The extent of separation of the cells at between junctions S R E PR receptors VEGFR1 and NRP1,

G E openings is governed by forces within villi VEGFR2 is active and button Intercellular junctions I MA transmitted to the lacteal wall by anchor-

A L junctions are converted into

OC ing filaments. Intake of dietary fat pro- zipper junctions. Zipper junctions

ON F motes chylomicron production and water VEGF-A high VEGFR2 active C ; Chylomicron uptake prreeventteeedd absorption by the intestinal epithelium Small intestine villus

C IE NCE that lead to higher interstitial pressure, S lymph flow, and chylomicron transport Capillary

U N I AN / through lacteals (5). Lacteal contractility, Lacteal O T AL . Lymphatic vessel S : V Department of Anatomy, University of California, San Francisco, CA 94143-0452, USA. Endothelial cell AP H I C Lymphatic endothelial cell nuclei G R Email: [email protected]

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DA_0810Perspectives.indd 551 8/8/18 11:46 AM INSIGHTS | PERSPECTIVES

addition to the gatekeeper function of cel- ESSAY lular junctions, chylomicron entry into lac- teals is controlled by the expression of the transcription factor pleomorphic adenoma The future of humans gene-like 2 (PLAGL2) in intestinal epithelial cells. Plagl2-deficient mice have impaired lacteal uptake of chylomicrons and die post- as model organisms natally from lack of fat absorption (13). The findings by Zhang et al. raise the A “human phenomic science” approach could question of how VEGF-A can have op- accelerate personalized medicine posite effects on lymphatics, where it re- duces permeability by promoting zipper junctions, and on blood vessels, where it By Garret FitzGerald1, David Botstein2, Complementary to large-scale, com- increases leakage by opening normally Robert Califf 3,4, Rory Collins5, Keith paratively lighter basal phenotype studies closed zipper junctions in endothelial Peters6, Nick Van Bruggen2, Dan Rader1 are deeper phenotyping studies of smaller cells. Although the basis of this dichotomy groups of individuals in settings where phe- is unclear, mechanistic insights come from en years ago, Nobel laureate Sydney notypic responses can be evoked under well- studies of Rho-associated protein kinases Brenner remarked, “We don’t have to controlled conditions (7). Such studies can (ROCKs). Pharmacologic inhibition of search for a model organism anymore. address and refine hypotheses generated ROCKs promote zippering of endothelial Because we are the model organisms” from data at scale, enabling investigators cell junctions and suppress chylomicron (1). Indeed, over the past decade, we interested in mechanisms and drug devel- Downloaded from uptake into lacteals. ROCK inhibitors also have deepened our understanding not opment to seek proof of concept in focused, reduce leakage in blood vessels (14). In Tonly of how the genomic blueprint for hu- prospective clinical trials. Deep phenotyping both cases, ROCK inhibitors increase the man biology manifests physical and chemical is ideally suited to elucidate gene function barrier function of endothelial cells by sta- characteristics (phenotype), but also of how and comes close to conducting a “human bilizing proteins at intercellular junctions. traits can change in response to the environ- gene knockout” study (8). The approach can These advances highlight the importance ment. A better grasp of the dynamic relation- http://science.sciencemag.org/ of button junctions in intestinal lacteals ship between genes and the environment for the uptake of dietary fat. Although but- may truly sharpen our ability to determine ton junctions were identified in lymphatics disease risk and response to therapy. A col- “Deep phenotyping … more than 10 years ago (3), the finding that lection of human phenotypic data, and its in- comes close to … a ‘human the junctions are essential for chylomicron tegration with “omic” information (genomic, uptake by lacteals and can be genetically proteomic, transcriptomic, epigenomic, mi- gene knockout’ study.” and pharmacologically regulated point to crobiomic, and metabolomic, among oth- new strategies for management of obesity. ers), along with remote-sensing data, could also connect the modifiable causes of com- However, understanding the mechanism provide extraordinary opportunities for dis- mon diseases to patients with rare diseases,

and consequences of the transformation of covery. A comprehensive “human phenomic enabling a better understanding of the patho- on August 9, 2018 button junctions into zippers is at an early science” approach could catalyze this effort physiology of common diseases. For example, stage. Zippering junctions in lymphatics through both large-scale “light” phenotyping detailed characterization of “outlier” patients could impair uptake of essential nutrients in studies and “deep” phenotyping studies per- can direct mechanistic interrogations of chylomicrons or have adverse effects by com- formed in smaller groups of individuals. common diseases at population scale. One promising fluid drainage and immune cell Data integration is already advancing example is the gene encoding proprotein trafficking. Nonetheless, the discovery that medicine at the individual patient level. The convertase subtilisin/kexin type 9 (PCSK9) the uptake of intestinal fat is determined by identification of unexpected disease associ- and cardiovascular disease (9). The car- the structure of junctions in lymphatic endo- ations with genes (2), the linkage of gene dioprotective phenotype that was revealed thelial cells opens new avenues for investi- variants to unanticipated human pheno- through light phenotyping at scale inferred gating the regulation of body weight. j types (3), and the use of Mendelian random- the validity of PCSK9 as a drug target; this ization (a method to estimate causal effects) was confirmed in deep phenotyping studies REFERENCES to predict biomarker validity (4) or drug re- and then in randomized clinical trials. An- 1. J. R. Casley-Smith, J. Cell Biol. 15, 259 (1962). sponse (5) are some examples. Efforts are other example is the autoimmune phenotype 2. W. O. Dobbins 3rd, E. L. Rollins, J. Ultrastruct. Res. 33, 29 (1970). underway to broaden the diversity of popu- of patients with atypical presentations of the 3. P. Baluk et al., J. Exp. Med. 204, 2349 (2007). lations studied and to establish standards myalgic encephalomyelitis/chronic fatigue 4. F. Zhang et al., Science 361, 599 (2018). for phenotyping (6), but the depth and qual- syndrome (10). Deep phenotyping revealed 5. P. R. Kvietys, D. N. Granger, Ann. N. Y. Acad. Sci. 1207 ity of characterization at scale are limited a mechanistic basis for a symptom complex (suppl. 1), E29 (2010). 6. K. Choe et al., J. Clin. Invest. 125, 4042 (2015). by cost and feasibility. that is sometimes miscategorized as an af- 7. C. Hagberg et al., Physiology 28, 125 (2013). fective disorder. Furthermore, the two ap- 8. M. R. Robciuc et al., Cell Metab. 23, 712 (2016). 1The Institute for Translational Medicine and Therapeutics, proaches to phenotyping might be integrated 9. L.-C. Yao et al., Am. J. Pathol. 180, 2561 (2012). The Perelman School of Medicine, University of Pennsylvania, to enable the identification of patients with 10. W. Zheng et al., Genes Dev. 28, 1592 (2014). 2 Philadelphia, PA 19104, USA. Calico Life Sciences, South rare diseases from large phenotypic datasets. 11. H. Nurmi et al., EMBO Mol. Med. 7, 1418 (2015). San Francisco, CA 94080, USA. 3Duke Forge, Duke University, 12. J. Bernier-Latmani et al., J. Clin. Invest. 125, 4572 (2015). Durham, NC 27710, USA. 4Verily Life Sciences, South San This could lead to more efficient recruitment 13. F. Van Dyck et al., Cell Metab. 6, 406 (2007). Francisco, CA 94080, USA. 5Clinical Trial Service Unit and for deep phenotyping studies. A major chal- 14. C. M. Mikelis et al., Nat. Commun. 6, 6725 (2015). Epidemiological Studies Unit, Nuffield Department of Population lenge will be harmonizing protocols for deep Health, University of Oxford, Oxford OX3 7LF, UK. 6University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, phenotyping. However, while moving toward 10.1126/science.aau5583 Cambridge CB2 2SP, UK. Email: [email protected] standardization, it may be possible to inte-

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